KASP primer combination suitable for lycopersicon esculentum DNA fingerprint database construction and application of KASP primer combination
A primer combination and primer set technology, which can be used in DNA/RNA fragmentation, recombinant DNA technology, determination/inspection of microorganisms, etc., and can solve problems such as rare and no special kits
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Embodiment 1
[0086] Embodiment 1, the screening of KASP primer combination
[0087] A large number of sequence analysis and functional analysis were performed on the tomato genome, and 48 SNP sites were screened (Table 2) (the physical coordinates refer to the tomato genome version 2.4, ftp: / / ftp.solgenomics.net / genomes / Solanum_lycopersicum / Heinz1706 / wgs / assembly / build_2.40 / ), and KASP primer design was carried out on it, and a large number of primers were screened among more than 1,000 pairs of primers designed to select primers with stable amplification effect and clear typing, and obtain the KASP primer combination constructed by the tomato DNA fingerprint library. Such as Table 3 (numbers in Table 3 correspond to those in Table 2) and figure 1 shown. Using the primers shown in Table 3 to carry out the results of the amplification detection of the materials shown in Table 1, see image 3 . Other primers (controls) in the screening process are used to carry out the results of amplifi...
Embodiment 2
[0095] Embodiment 2, construction and application of tomato DNA fingerprint library
[0096] 1. Construction of Tomato DNA Fingerprint Library
[0097] 1. Genomic DNA of 96 young leaves of tomato (Table 1) to be tested was extracted by CTAB method.
[0098] 2. Use 48 sets of primers in Table 3 to carry out PCR amplification on 96 tomato samples to be tested (48 sets of primers are in the same reaction system), and detect the genotypes of the tomato to be tested at 48 SNP sites. Fluorescent quantitative PCR instrument AB-Q6 Flex can be used for detection or the ArrayTape platform of Douglas Scientific company can be used for detection.
[0099]The reaction system (5 μL) when using the fluorescent quantitative PCR instrument AB-Q6 Flex (Thermo Fisher Scientific (China) Co., Ltd.) detection is: Genomic DNA (50ng / μL) 1.0 μL + primer mixture 0.7 μL (in the primer mixture The concentration of each primer in the set of primers in Example 1 is 100 pmol / L) + 2×KASP Mix (Aiji Analysis...
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